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OPEN
Received: 4 July 2019
Accepted: 20 August 2019
Published: xx xx xxxx
Scientific Reports |
Childhood iron deficiency anemia
leads to recurrent respiratory tract
infections and gastroenteritis
Jayaweera Arachchige Asela Sampath Jayaweera   1, Mohammed Reyes2 &
Anpalaham Joseph1
Anemia affects approximately 30% of children all over the world. Acute respiratory tract infections
(ARTI), urinary tract infections (UTI) and gastroenteritis (GE) are common infectious entities in children.
Here, we assessed the association between anemia and development of recurrent ARTI, UTI, and GE
in children. This was a case-control study in hospitalized 2–5 years old children in Professorial Pediatric
Unit at Teaching Hospital Anuradhapura, Sri Lanka. An 18-month follow up was done to assess the
risk factors for the development of recurrent ARTI, GE, UTI, and control presented without infections.
Further, 6-month follow up done after 3-month iron supplementation to assess the occurrence of
recurrences. Blood Hb concentration was measured using Drabking’s reagent. Logistic regression was
used to find the risk factors for the development of recurrences. In ARTI, 121/165 (73.3%), GE, 88/124
(71%), UTI 46/96 (47.9%) and control 40/100 (40%) were having anemia. Initial ARTI group, recurrent
ARTI was 24 (14.5%, p = 0.03); initial GE group: recurrent GE was 14 (11.3%, p = 0.03), recurrent
ARTI was 11 (8.9%, p = 0.04); initial UTI group, development of; recurrent UTI was 8 (8.3%, p = 0.04);
control, recurrent ARTI was 11 (11%, p = 0.03). Following 3-month iron supplementation reduction
of recurrences was significant: initial ARTI recurrent ARTI in 90%, recurrent GE in 77.7%; initial GE
recurrent GE in 83.3%, recurrent ARTI in 80%; initial UTI recurrent ARTI in 71.4% and control recurrent
ARTI in 88.8%. Iron deficiency is a major type of anemia and anemic children are more prone to develop
recurrent ARTI and GE. Once iron deficiency being corrected the rate of recurrent ARTI and GE was
reduced. This would be a boost for policy developers to implement strategies at the community level to
prevent iron deficiency in children to reduce ARTI and GE recurrences.
Acute infective episodes in children are quite common and are associated with high morbidity and mortality1.
Acute respiratory tract infections (ARTI), urinary tract infections (UTI) and gastroenteritis (GE) are such com-
mon infectious entities2–5. In the globe, infections following bacteria and viruses play a significant role while
parasites and fungi are emerging and threatening6,7.
The disease burden following childhood ARTI is greater than that of any other cause of disease1. In 2014, 18%
of mortality for children younger than 5 years of age was caused by ARTI while the diarrheal disease is the next
greatest8,9. UTI in children <5 years of age the associated burden is 4%. Further the overall conclusion one out of
20 girls and one out of 50 boys will have a UTI by the age of 5 years, with a predominance of boys during the neo-
natal period and early infancy10. Overall burden following these 3 major acute childhood infections is substantial
thus impact on the globe and the country economy is enormous11.
Following entry of microorganism to the organ vicinity several factors concurrently contribute for the devel-
opment of the infection. When considering host-parasite interface nutritional status of the host is one of the key
contributory factors for invasion and development of infections12–14. Hemoglobin (Hb) concentration is a param-
eter that reflects the chronic nutritional status and also blood oxygen carrying capacity15,16. Children are at a rapid
growth state thus demand the nutrition is enormous17. Simultaneously, the tendency to develop under-nutrition
is also high. In such instances, the risk for development of infection is high and the vicious cycle continues leads
to poor-nutrition18.
1
Department of Microbiology, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri
Lanka. 2Department of Pediatrics, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura,
Sri Lanka. Correspondence and requests for materials should be addressed to J.A.A.S.J. (email: jaas071@gmail.com)
(2019) 9:12637 | https://doi.org/10.1038/s41598-019-49122-z 1
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Anemia is affecting approximately 30% of children all over the world19,20. Several factors are contributed to
anemia. In childhood, nutritional anemia including iron, vitamin B-12, and folate deficiency is the commonest21.
In addition to the nutritional, hereditary type of anemia including thalassemia, sickle cell anemia and aplastic
variety following bone marrow suppression is observed22. Irrespective of the etiology following anemia, child
suffers fatigability and its negative effect on growth is great. Iron deficiency anemia in children occurs most
frequently between the age of 6 months and 3 years, the period of age when repeated infections occur23. Anemia
associated lower ARTI occurs more commonly in children than in adults.
Recurrent ARTI and UTI are common in children and following development of recurrences the associated
burden would worsen24,25. Recurrent infective episodes invariably lead to undernutrition26. Perhaps, anemia is
a well-known risk factor for recurrent infective episodes27. In addition, studies on hemoglobin level and devel-
opment of multiple episodes of otitis media in children been discussed28. In a cross-sectional case-control study
anemia and occurrence of acute gastroenteritis also been discussed among children in Gaza strip29. In contrast,
anemia and development of recurrent UTI are not been well described in the literature. Another hand if a child
develops recurrent infections possibility of immunodeficiency need to be excluded30.
Irrespective to the etiology of anemia, the relation between low hemoglobin (Hb) level and occurrence of
infections has not been fully evaluated, and only a few reports are available31. This study would assess the Hb
status and development of acute as well as recurrent ARTI, UTI, and GE in children.

Method
This was a case-control study in hospitalized 2–5 years old children with ARTI, UTI, and GE over March 2014
to August 2014. To participate in the study informed written consent obtained from the legal guardians/parents.
As a control, children presented to the outpatient department to seek treatment for traumatic surgical cases
who were having past 6-month period free of any acute or chronic infections were included. Children having
pulmonary, cardiac, gastrointestinal and urogenital structural and functional anomalies were excluded from the
study. Patients who undergo repeated blood transfusions were excluded from the study. Further, children with
known immunodeficiencies were also excluded from the study. The study was performed at the pediatric ward,
Professorial Unit, Teaching Hospital Anuradhapura, Sri Lanka. Participants including controls were followed up
18 months to assess the occurrence of recurrent ARTI, GE and UTI and the risk factors. This was done following
weekly telephone conversations with the guardians. In each episode they were asked to admit to the Professorial
Unit, Teaching Hospital Anuradhapura, Sri Lanka. Further, a period of 6 months was followed up following iron
supplementation (3-month period) to assess the development of recurrent infections. They were followed up in
similar manner. All methods and protocols were performed in accordance with the relevant approved guidelines
and regulations.
ARTI cases with Severe Acute Respiratory Illness (SARI) defined by WHO were included. A child has a fever
with dysuria (crying while micturition) and or hematuria was included as UTI. Sudden onset of diarrhea and/
or vomiting, usually three or more bouts of diarrhea or vomiting were taken as GE. A hundred patients in age
2–5 years who visited the outpatient department for surgical problems were taken as a control. Definition of
recurrent ARTI is arbitrary, too generic, restrictive and for our study: recurrent infectious rhinitis as more than
five episodes per year and recurrent pharyngitis or tonsillitis more than three episodes within 12 months. For the
lower respiratory tract, we have taken ≥3 episodes per 12 months. Similarly, for recurrent GE we have taken ≥3
episodes per 12 months while for the clarity cases with chronic diarrhea were excluded. Recurrent urinary tract
infection (UTI) refers to ≥2 infections in six months or ≥3 infections in one year. For anemia when hemoglobin
level was considered age-specific 13- ≤ 24 months [mean 12.0 g/dL (−2SD: 11.0 g/dL)] and 25- ≤ 60 months
[mean 12.5 g/dL (−2SD: 11.5 g/dL)] *Brian Yang Merritt’s Haemoglobin concentration (http://emedicine.med-
scape.com/article/2085614-overview) below 2 standard deviation (SD)32.
Children with ARTI nasopharyngeal aspirates (NPAs) collected with the help of recommended mucus extrac-
tor by the pediatrician/prior trained medical doctor (research candidate). Indirect immunofluorescence assay was
™
performed by DAKO IMAGEN (United Kingdom)33, respiratory screening reagents for 8 respiratory viruses
and viral typing was done for each of RSV, adeno, parainfluenza 1, 2 & 3, influenza A & B and Human metapneu-
™
movirus (hMPV) viruses using monoclonal antibodies DAKO IMAGEN (United Kingdom). Children with
UTI were having significant (single isolate or mixed > 105) and culture growth from clean caught urine was taken
as having bacterial UTI. In GE stool full report was having a significant number of pus cells considered. The col-
lected human fecal samples were tested in duplicate for Group A rotavirus and Adenovirus using a commercially
available qualitative enzyme immunoassay (ProSpectTM Rotavirus Microplate Assay manufactured by Oxoid Ltd,
UK and ProSpectTM Adenovirus Microplate Assay manufactured by Oxoid Ltd, UK, respectively), following the
manufacturer’s instructions.
Each stool was inoculated into selenite brilliant green sulfa enrichment broth (Oxoid Ltd, Basingstoke, UK)
at 37 °C for 18 h, and was then plated onto Salmonella-Shigella agar (Oxoid Ltd, Basingstoke, UK) and xylose
lysine deoxycholate agar (Oxoid Ltd, Basingstoke, UK) to detect non typhoidal salmonellosis and Shigella spp.
strains, after an 18 h incubation at 37 °C. The suspicious colony was plated onto CHROMagar Salmonella™
medium (CHROMagar, Paris, France) and cultivated at 37 °C for 18 h. Each stool sample was directly inocu-
lated onto alkaline peptone water (Oxoid Ltd, Basingstoke, UK) at 37 °C for 18 h to examine for Vibrio cholera,
Vibrio parahaemolyticus, Aeromonas spp., and Plesiomonas spp., and was then plated onto thiosulfate-citrate-bile
salts-sucrose agar (Oxoid Ltd, Basingstoke, UK) at 37 °C for 18 h. Suspicious colonies were selected to conduct the
oxidase experiment. If the oxidase test resulted in a positive reading, the systematic biochemical identification for
these suspicious colonies was confirmed.
Stool iodine staining, wet smear, and microscopy were performed to assess amoebic cysts, oocytes, and other
helminth oocytes and larvae. In addition to that fecal reducing substances, the level was taken to exclude lactose
intolerance and malabsorption syndromes. Blood Hb concentration from all participants was measured using

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Drabking’s reagent using a spectrophotometer. Blood picture analysis and serum ferritin levels were measured to
define the etiology for anemia.
An investigator administered questionnaire was used to collect patients’ demography, nutritional status, clin-
ical presentation, and past medical history. For iron deficiency anemia following a period of 3-month of oral iron
supplementation (weight/based) the subjects were further followed up over 6-month to observe the development
of recurrent ARTI, GE and UTI. Children with hemoglobin 9–10 g/dL were supplemented with 60–120 mg of
iron. Hemoglobin concentration, blood picture (normochromic and normocytic) and assessment of serum ferri-
tin level was done to confirm the cure of iron deficiency anemia.
®
Data obtained were double entered into a spreadsheet database prepared with Microsoft Excel and compared
and cleaned for wrong entries. Statistical analysis was done using SAS version 9.1 (SAS, 2005, New Jersey)34.
Association of each of the categorical variable with response variable was assessed by Chi-square test. Variables
showing statistically significant association in univariate analysis with the outcome variable were considered as
a risk factor. Only those variables were subjected to multivariate analysis. Logistic regression method was used
to find the risk factor for the development of recurrent ARTI, UTI, and GE. In multivariate analysis, variables
showing P < 0.05 were considered to be statistically significant. Continuous variables were expressed as a measure
of central tendency.

Ethics approval and consent to participate.  Ethical approval for all experimental protocol/s were
approved by ethical review and publication committee, University of Peradeniya, Sri Lanka and to participate in
the study informed written consent obtained from the legal guardians/parents.

Results
Over the period of the initial 18 months, children with clinically suspected 165 cases of ARTI, 124 cases of GE,
96 cases of UTI and 100 control were enrolled in the study. Children with suspected ARTI, 65 (39.3%) cases of
viral ARTI was detected based on IFA, mean age of presentation was 2.4 ± 0.5 years and mean hospital stay was
4 ± 2 days while 58% were males and 42% females. Children with GE, 62 (50%) viral, 12 (10%) bacterial cases
detected based on viral ELISA and stool bacterial cultures respectively. The mean age of presentation of GE was
2.5 ± 0.8 years and mean hospital stay was 5 ± 2.5 days while 57% were males and 43% females. Children with
UTI, 52 (54.1%) had culture-positive UTI mean age of presentation was 2.45 ± 0.7 years and mean hospital stay
was 3 ± 2.4 days while 55% were males and 45% females. In control, 52% were males and 48% were females. In all
including the control, male predominance (p = 0.03) with no significant difference between mean age and mean
hospital stay was detected.
Out of 165 children with ARTI, 121 were anemics (73.3%). Eighty-eight children out of 124 with GE and
(71%) and 46 out of 96 children with UTI were having anemia (47.9%). In control subjects, 40 of them were
having anemia (40%). When compared the diseased groups and the control, children with ARTI and GE were
having anemia significantly (p = 0.03 and 0.04 respectively) whiles children with UTI, anemia was not significant
(P > 0.05) (Table 1).
When considering anemia, in initial ARTI group 121 (73.3%) of them were found to have iron deficiency
anemia, 2 of them with megaloblastic anemia and 1 with asymptomatic sickle SD disease. Whiles in initial GE
group 86 (71.0%) were found to have iron deficiency anemia, 2 of them with megaloblastic anemia. In UTI group
44 (47.9%) were found to have iron deficiency anemia and 2 of them were thalassemia trait. Further in control
38 (95%) were found to have iron deficiency anemia and 2 of them were thalassemia traits. In the control group,
35 (35%) found to have iron deficiency anemia. We have excluded patients having repeated blood transfusions.
Therefore, patients having thalassemia major and other hemoglobinopathies demanding blood transfusions were
not considered for the analysis. Based on blood picture analysis, iron deficiency anemia was further confirmed by
serum ferritin assay. In initial ARTI patients, serum ferritin level in patients with anemia was 4.5 ± 0.3 ng/ml. In
initial GE patients, it was 4.5 ± 0.4 ng/ml, in initial UTI patients it was 4.5 ± 0.35 ng/ml and in control with ane-
mia it was 4.5 ± 0.3 ng/ml. It was significantly (p = 0.03) below the age-specific lower limit of the reference range
(<6 ng/ml in both male and female <5 ng/ml years of age). In between anemic children in ARTI, GE, UTI groups
and control the serum ferritin values were not significantly differed (p > 0.05). Further, no peripheral stigmata of
iron deficiency anemia were observed. All of the subjects were on anti-helminth treatment once in six months.
Further patients with GE, non-of them were having amoebic oocytes, cysts and any oocytes related to soil and
non-soil inhabiting helminths in wet stool mounts. Also, on iodine staining, all tested diarrheal stool was negative
for parasite cyst or ova.
Anemia was a risk factor for the development of ARTI with an odds ratio of 3.08 with 95% interval confidence
of 2.03–4.80 (P = 0.004) Further anemia was a risk factor for the development of GE with an odds Ratio of 2.98
with 95% interval confidence of 1.93–4.40 (P = 0.01). In UTI group anemia was not either risk or a protective
factor as the odds ratio of 1.03 (p = 0.09) with 95% interval confidence of 0.78–1.40.
Over the 18 months follow up period development of recurrent ARTI, GE and UTI in study groups and the
control as follows. Among initial ARTI group, development of recurrent ARTI was 24 (14.5%, p = 0.03); recurrent
GE was 11 (6.5%, p = 0.06) and recurrent UTI was 0. Among initial GE group, development of; recurrent GE was
14 (11.3%, p = 0.03), recurrent ARTI was 11 (8.9%, p = 0.04) and recurrent UTI was 4 (3.2%, p = 0.07). Among
initial UTI group, development of; recurrent UTI was 8 (8.3%, p = 0.04), recurrent ARTI was 4 (4.1%, p = 0.06)
and recurrent GE was 0. Among control, development of; recurrent ARTI was 11 (11%, p = 0.03), recurrent
GE was 6 (6%, p = 0.06) and recurrent UTI was 2 (2%, p = 0.08). For the risk factor analysis, only significant
(p < 0.05) recurrent infections among study groups was included (Table 2).
Among initial ARTI group for the development of recurrent ARTI; male sex, height for age <−2 SD and Hb%
<11 g/dL were significant risk factors whiles in initial GE group for the development of recurrent GE; height for
age <−2 SD, Hb% <11 g/dL and not washing hands prior to handling of child were significant risk factors. Also,

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Control P value and

Initial group (n) ARTI (165) GE (124) UTI (96) (100) comments
Percentage of Iron deficiency anemia (%) 121 (73.3%) 86 (71.0%) 44 (47.9%) 40 (40%) 0.03
ARTI GE GE ARTI ARTI ARTI
Recurrent infections among anemics (%) 20 9 12 10 7 9 —
(16.5%) (7.4%) (14%) (11.6%) (16%) (22.5%)
ARTI GE GE ARTI ARTI ARTI
Recurrent infections among anemics (%) 4 2 2 1 1 2 —
(2.4%) (1.6%) (1.6%) (0.8%) (1%) (2%)
Initial Hb (g/dl) 9.6 ± 0.8 9.5 ± 0.7 9.7 ± 0.8 9.6 ± 0.9 —
Initial serum ferritin (ng/ml) 4.5 ± 1.2 4.5 ± 1.1 4.5 ± 1.2 5.5 ± 0.3 —
3-month Iron supplementation
Initial group ARTI GE UTI Control
At 3-month Hb (g/dl) 11.3 ± 0.2* 11.6 ± 0.4* 11.4 ± 0.3* 11.9 ± 0.3* 0.03*
** ** ** **
serum ferritin (ng/ml) 12.6 ± 0.8 12.6 ± 0.4 12.6 ± 0.8 12.2 ± 0.7 0.02**
Follow up- 6 months
ARTI GE GE ARTI ARTI ARTI
Recurrent infection 2 2 2 2 2 1 —
(1.6%) (1.6%) (2.3%) (2.3%) (4.5%) (2.5%)
At 6-month Hb (g/dl) 11.6 ± 0.4 12.1 ± 0.6 12.4 ± 0.3 11.9 ± 0.2 —
ARTI GE GE ARTI ARTI ARTI
Reduction of recurrences over 6 month (%) 0.03
90 77.7 83.3 80 71.4 88.8

Table 1.  Details of recurrent infections before, after 3 month of iron supplementation and 6 months follow up.
ARTI- acute respiratory tract infections, GE- gastro-enteritis, UTI- Urinary tract infections. P < 0.05 taken as
significant. -: not significant.

Initial disease ARTI GE UTI Control

Significant recurrent infection in above ARTI GE ARTI UTI ARTI P value and
disease groups OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) comments
Risk factors
Sex
   Male 1.6 (1. 5–1.8) — — — 0.03
   Female — — 1.6 (1.4–1.8) — 0.04
Height for age
(<−2 SD)
2.6
®
(2.1–3.1 )
1.6 (1.4–1.8)* 1.6 (1.4–1.8) µ
— 1.6 (1.4–1.8) ®
0.03 , 0.04*,
0.04µ, 0.04
5.2 0.03®, 0.04*,
(4.5–5.9)®
Hb (<11 g/dL) 3.6 (3.1–4.1)* 4.5 (4.0–4.9)µ — 4.2 (3.4–4.9) µ
0.04 , 0.04
Constipation — — — 2.6 (2.2–3.0) — 0.04
Water intake <1l — — — 1.7 (1.4–2.0) — 0.04
Hand washing prior to handling of child — 1.5 (1.3–1.7)µ 1.8 (1.3–2.2) — — 0.03µ, 0.04
National program of immunization coverage 100% 100% 100% 100% 100% —
Anti-helminth use-every 6 months 86% 88% 84% 90% 88% —

Table 2.  Factors associated with development of recurrent acute respiratory tract infection (ARTI),
gastroenteritis (GE) and urinary tract infection (UTI) among followed up ARTI, GE, UTI and control groups.
ARTI- acute respiratory tract infections, GE- gastro-enteritis, UTI- Urinary tract infections, LSCS- lower
segmental caesarian section, OR- odds ratio, SD- standard deviation. Only significant factors were included.
P < 0.05 taken as significant. -: not significant.

in initial GE group for the occurrence of recurrent ARTI; height for age <−2 SD, Hb% <11 g/dL and not washing
hands prior to handling of the child were significant risk factors. Further, among initial UTI group for the occur-
rence of recurrent UTI; female sex, constipation, low water intake, and structural malformations were significant
risk factors. In addition, among control group for the occurrence of recurrent ARTI; height for age <−2 SD and
Hb% <11 g/dL were significant risk factors.
None of these factors were not associated significantly (p > 0.05) with recurrences in test subjects and the
control. Birth weight (<2500 g), maturity (<36 weeks period of amenorrhea), mode of delivery (normal vaginal
or caesarian section), weight for age (<−2 SD), type of drinking water source (tap, tank or spring), use of boiled
cooled water, having daily bath and frequent (>2/day) body wash, exclusive breast feeding in first 4 months,
family monthly income <30,000 rupees, mothers’/caregivers’ education (only up to primary level), proper
waste disposal, birth order (3rd or more in order) and day care attendee, having congenital anomalies including
cyanotic heart diseases, cystic fibrosis, structural anomalies in gastro-intestinal tract familial syndromes, having
gastro-esophageal reflux and bronchial asthma.

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Following a period of 3-month of iron supplementation (60–120 mg daily), the subjects were further followed
up period of 6-month to observe the development of recurrent ARTI and GE. Meanwhile, advice on hand hygiene
was also given. Recurrent infection among anemics as follow: initial ARTI subjects recurrent ARTI was detected
in 20 (16.5%), recurrent GE was detected in 9 (7.4%); initial GE subjects recurrent GE was detected in 12 (10%),
recurrent ARTI was detected in 10 (12%); initial UTI subjects development of recurrent ARTI was detected in
7 (16%) and control subjects development of recurrent ARTI was detected in 9 (25%). Following 3-month oral
iron supplementation hemoglobin concentration was increased significantly and (mean ± SD) mg/Dl as follows:
initial ARTI- 11.3 ± 0.2 (p = 0.03), GE- 11.6 ± 0.4 (P = 0.03), UTI- 11.4 ± 0.3 (P = 0.03) and controls- 11.9 ± 0.3
(p = 0.03). serum ferritin was increased significantly and (mean ± SD) ng/ml as follows: initial ARTI- 12.6 ± 0.8
(p = 0.02), GE- 12.6 ± 0.4 (P = 0.02), UTI- 12.6 ± 0.8 (P = 0.02) and controls- 12.2 ± 0.7 (p = 0.02). Recurrent
infection rates were reduced significantly.
Initial ARTI subjects recurrent ARTI was detected in 2 (1.6%), recurrent GE was detected in 2 (1.6%); ini-
tial GE subjects recurrent GE was detected in 2 (10%), recurrent ARTI was detected in 2 (12%); initial UTI
subjects recurrent ARTI was detected in 2 (16%) and control subjects recurrent ARTI was detected in 1 (25%).
Reduction of recurrences over 6-month follow up as follows: Initial ARTI subjects recurrent ARTI was reduced
90%, recurrent GE was reduced in 77.7%; initial GE subjects recurrent GE was reduced in 83.3%, recurrent ARTI
was reduced in 80%; initial UTI subjects recurrent ARTI was reduced in 71.4% and control subjects recurrent
ARTI was reduced in 88.8% (Table 1).
In subjects with normal hemoglobin the recurrent infections as follows. In initial ARTI group, development
of; recurrent ARTI was 4 (2.4%, p = 0.09), recurrent GE was 2 (1.6%, p = 0.07) and recurrent UTI was 0. Among
initial GE group, development of; recurrent GE was 2 (1.6%, p = 0.08), recurrent ARTI was 1 (0.8%, p = 0.1)
and recurrent UTI was 0 (0%, p = 0.07). Among initial UTI group, development of; recurrent UTI was 1 (1%,
p = 0.14), recurrent ARTI was 0 (0%, p = 0.06) and recurrent GE was 0. Among control, development of; recur-
rent ARTI was 2 (2%, p = 0.06), recurrent GE was 1 (1%, p = 0.07) and recurrent UTI was 1 (1%, p = 0.07). All
were not significant (p > 0.05).
In initial ARTI patients out of 121 children with anemia viral etiology was detected in 52 (43%) cases.
Respiratory syncytial virus (RSV) was commonly detected in the virus (60%) in children with ARTI. In initial GE
patients out of 86, children with anemia etiology (viral-44, bacterial-2) were detected in 56 (65%) cases. Rotavirus
(57%) was commonly detected in children with GE. In initial UTI patients out of 44 children with anemia, etiol-
ogy was detected in 36 (82%) cases. E. coli (64%) was commonly detected in children with UTI. Etiology of initial
ARTI, GE and UTI groups and subsequent recurrences over the follow-up period following oral iron supplemen-
tation was displayed on Table 3. Recurrences following RSV was common in initial ARTI as well as other groups.
Also, recurrences following RV was common in initial GE as well as other groups. The overall rate of RSV and RV
recurrence in all groups have significantly reduced following oral iron supplementation (Table 3).
Further, primary or secondary immunodeficiency was not detected in any of the subjects including the
control.

Discussion
Childhood nutritional anemia would reflect the status of chronic malnutrition15,16. The world health organization
estimates that globally around 293 million young children suffer from anemia, among which 50% are estimated
to be attributable to iron deficiency. Iron deficiency anemia can be present at early age and also in well-nourished
children21. Iron deficiency is one of the most common micronutrient deficiencies in the world22.
Children are vulnerable for various infections specially ARTI, UTI and GE10,11. Such infections could be asso-
ciated with a low level of immunity. Frequent exposure and low level of hygienic practices are associated with
recurrences11,35. Once recurrent infections are associated with childhood under-nutrition the outcome would
worsen often ended up with frequent infections in early life. This is a modifiable risk factor for the development
of infections16,18. While adherence to hygienic practices, consumption of nutritionally adequate diet would lead
to alleviating the burden16,35.
In our study, the risk of childhood ARTI was significantly associated with iron deficiency anemia. Blood iron
deficiency is a risk factor for the development of recurrent ARTIs25,26. Mourad et al. and Ramakrishnan et al.
shows that iron deficiency anemic children were two times and five times more susceptible to lower respiratory
tract infection compared to the control group, respectively31,36. Adequate iron is important for proliferation and
maturation of immune cells, particularly lymphocytes, for generation of specific response to infection37. Further
the observed risk could be due to low oxygen carrying capacity in pulmonary vasculature and parenchyma lead-
ing to the low level of protectively towards invading pathogens. Among viral ARTI, recurrence was common fol-
lowing RSV. Is the most prevalent virus among childhood ARTI and frequent exposure would lead to recurrences.
RSV is considered one of the earliest stimuli for recurrent wheezing in children38. The supplementation of iron in
healthy childhood community has reduced upper respiratory tract infections significantly38–40.
Iron deficiency is a risk factor for the development of GE. Also, GE would be associated with malnutrition.
Since gastric epithelium having a high turnover rate, it requires well nourish status for maintenance of mucosal
integrity and absorptive capability41,42. Further cumulatively low level of nutrition leads to low immunity.
Rotavirus was detected as commonest etiology for childhood GE as well as recurrent GE. Frequent exposure
would lead to recurrences43. In here, the stool was tested only in single sample helminth infestation cannot be
excluded. Continuous use of anti-helminth thus leads to the low incidence of helminth infestation.
Interestingly, anemia is neither a risk factor nor a protective factor for the development of childhood UTI.
Level of immunity perhaps with low in mal-nutrition but low level of hydration is key to the development of UTI.
In addition to that structural malformations and anatomical anomalies act simultaneously for the acquisition of
childhood UTI44.

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Initial group
anemics (n) ARTI (121) GE (86) UTI (44) Control (35)
Bacterial etiology -36
Viral etiology -52 (43%) (82%)
RSV- 31 (26%), PIV1-3 (2.5%), PIV Etiology detected -56 (65%) Escherichia coli- 23 (53%),
2–3 (2.5%), AV-2 (1.6%), hMPV-4 RV-32 (37%), AV(g)- 12 (14%), Klebsiella pneumoniae-6
Etiology —
(3.3%), Influenza A- 6 (5%), Shigella sonnei-2 (2.3%) (14%), Staphylococcus
Influenza B- 3 (2.5%), No etiology -30 (35%) aureus- 2(4.5%), proteus
No etiology 69 (57%) sp.-3(6.7%)
No etiology -8 (18%)
Significant
ARTI GE GE ARTI ARTI ARTI
recurrent
20 9 12 10 7 9
infections
(16.5%) (7.4%) (10%) (12%) (16%) (25%)
among anemics
RSV- 18 (90%),
RV-8 (89%), RV-10 (83%), RSV-8 (89%)
Etiology Influenza A- 2 RSV-10 (100%) RSV-7 (100%)
AV- 1 (11%) AV- 2 (17%) AV-1 (11%)
(10%)
3-month Iron supplementation
ARTI GE UTI Control
Recurrent
infection at 6 ARTI GE GE ARTI ARTI ARTI
months follow 2 2 2 2 2 1
up
Etiology RSV-2 (100%) RV-2 (100%) RV-2 (100%) RSV-2 (100%) RSV-2 (100%) RSV-1 (100%)

Table 3.  Etiology of recurrent infections before, after 3 month of iron supplementation and 6 months follow
up. ARTI- acute respiratory tract infections, UTI- urinary tract infection, GE-gastro-enteritis, RSV-Respiratory
syncytial virus, AV- Adenovirus, %), PIV1-parainfluenza virus-1, PIV 2- parainfluenza virus-2, hMPV-human
Metapneumovirus, AV(g)- adenovirus causing gastro-enteritis.

Following iron supplementation hemoglobin concentration rose, became normal for age and sex. The increase
in serum ferritin reflects the correction of iron storages. Over the follow-up period, recurrent ARTI and GE
among tested groups were significantly reduced thus indicating replenishing iron in blood plays a significant
protective role in childhood recurrent ARTI and GE. World Health Organization advice to supplement iron to
prevent iron deficiency in the population to minimize infections37,45,46. Meanwhile, advice on hand hygiene and
sanitary practices were also given as health education.
Sri Lanka comprises well spread public health facilities with great awareness but the majority of children suffer
anemia. Although we did not collect data on dietary intake of infants (except breast milk intake and introduction
of solid/semisolid foods), evidence from other studies from rural Sri Lanka suggests that dietary diversity is low
and might also be responsible for anemia47,48. Poverty will be a key factor23,46. Also feeding mal-practices and lack
of knowledge on nutritive food materials would aggravate it. It is important that implementation of ground-level
education on nutrition and supplementation of macro and micro-nutrients on a regular basis. This would reduce
the level of childhood infections and associated burden.
Here, we haven’t measured the level of adherence to hand hygiene and sanitary practices. This could be a con-
founding factor on the reduction of infections and the recurrences.

Conclusion
Children are vulnerable for developing various infections specially ARTI, UTI and GE. Iron deficient children
are more prone to develop recurrent ARTI, GE and iron deficiency anemia would worsen the associated burden.
Once iron deficiency being corrected the rate of recurrent ARTI and GE was reduced. This would be a boost for
policy developers to implement strategies at the community level to prevent iron deficiency in children to reduce
ARTI and GE recurrences.

Data Availability
The datasets used and analyzed in the current study are available from the corresponding author on reasonable
request.

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Author Contributions
J.A.A.S.J. and M.L.M.R. designed the study and all authors participated in data analysis. J.A.A.S.J. and A.J. carried
out the lab work. J.A.A.S.J. drafted the manuscript, and the final manuscript was read and approved by all authors.

Additional Information
Competing Interests: The authors declare no competing interests.
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